Detection of Enterotoxigenic Psychrotrophic Presumptive Bacillus cereus and Cereulide Producers in Food Products and Ingredients

In the last decade, foodborne outbreaks and individual cases caused by bacterial toxins showed an increasing trend. The major contributors are enterotoxins and cereulide produced by Bacillus cereus, which can cause a diarrheal and emetic form of the disease, respectively. These diseases usually induce relatively mild symptoms; however, fatal cases have been reported. With the aim to detected potential toxin producers that are able to grow at refrigerator temperatures and subsequently produce cereulide, we screened the prevalence of enterotoxin and cereulide toxin gene carriers and the psychrotrophic capacity of presumptive B. cereus obtained from 250 food products (cereal products, including rice and seeds/pulses, dairy-based products, dried vegetables, mixed food, herbs, and spices). Of tested food products, 226/250 (90.4%) contained presumptive B. cereus, which communities were further tested for the presence of nheA, hblA, cytK-1, and ces genes. Food products were mainly contaminated with the nheA B. cereus carriers (77.9%), followed by hblA (64.8%), ces (23.2%), and cytK-1 (4.4%). Toxigenic B. cereus communities were further subjected to refrigerated (4 and 7 °C) and mild abuse temperatures (10 °C). Overall, 77% (94/121), 86% (104/121), and 100% (121/121) were able to grow at 4, 7, and 10 °C, respectively. Enterotoxin and cereulide potential producers were detected in 81% of psychrotrophic presumptive B. cereus. Toxin encoding genes nheA, hblA, and ces gene were found in 77.2, 55, and 11.7% of tested samples, respectively. None of the psychrotrophic presumptive B. cereus were carriers of the cytotoxin K-1 encoding gene (cytK-1). Nearly half of emetic psychrotrophic B. cereus were able to produce cereulide in optimal conditions. At 4 °C none of the examined psychrotrophs produced cereulide. The results of this research highlight the high prevalence of B. cereus and the omnipresence of toxin gene harboring presumptive B. cereus that can grow at refrigerator temperatures, with a focus on cereulide producers.

In the last decade, B. cereus food poisonings outbreaks and individual cases showed an increasing trend, and in 2020, as the most frequently reported cause of food poisoning outbreaks in the EU [24][25][26][27][28]. Considering that B. cereus might induce diseases with fatal consequences, understanding B. cereus prevalence in food products and its ability to grow at refrigerator temperatures is an essential prerequisite to assure food safety. Therefore, the aim of this study was to estimate the prevalence of toxigenic B. cereus organisms among selected food products and ingredients, as well as the growth properties of B. cereus grown at refrigerator (4 and 7 • C) and mild abuse temperatures (10 • C). Additionally, the ability of psychrotrophic ces-positive presumptive B. cereus to produce cereulide at 37 • C and 4 • C was analyzed.

Distribution of Toxin Genes among Presumptive B. cereus Organisms Obtained from Food Products
B. cereus is one of the most common causal agents of foodborne diseases among bacterial toxin producers. Reports published by the European Food Safety Agency (EFSA) and European Center for Disease Control (ECDC) demonstrated that many food types were vehicles in strong-evidenced foodborne outbreaks [1,26,27,34,35]. In this study, we collected 250 food products and ingredients from the Belgian, Dutch and Serbian retail markets with the aim to estimate the prevalence of contaminated food products by the toxigenic B. cereus, toxigenic profiles of psychrotrophically grown presumptive B. cereus communities, and their ability to produce cereulide. In total, 226/250 (90.4%) food products contained presumptive B. cereus, which we further tested for the presence of nheA, hblA, cytK-1, and ces genes. Overall, selected food products were mainly contaminated with the nheA positive B. cereus carriers, found in 77.9% of the samples (176/250). Typically, nhe positive strains are the most prevalent among B. cereus food isolates [36][37][38][39][40]. However, the prevalence of the nhe gene among B. cereus food isolates reported in literature depends on the food type, as some studies indicated that the hbl gene was the most prevalent [41,42]. In our study, data showed that hblA positive presumptive B. cereus were found in 64.8% (162/250) of collected food samples. Also, Wijnands et al. [37] reported that hbl was detected in approximately 66% of the food isolates. Something higher proportion of Hbl-producing strains (766/1022), was reported by Berthold-Pluta et al. [38] in products collected from the Polish market (74.9%). Biesta-Peters et al. [39] found that nearly half of the food isolates contained the hbl genes.
Further, in our study, 23.2% (58/250) of examined food products were contaminated with emetic B. cereus (Table 1). Similarly, around 17% of food isolates obtained from the Dutch market were classified as emetic strains [39]. However, not all emetic strains were able to produce cereulide, indicating that toxin encoding genes do not always express their pathogenic potential. In our study, we observed that among psychrotrophic ces-positive presumptive B. cereus producers 5 out of 11 were able to produce cereulide (Section 2.4). Further, 10 out of 250 products were contaminated with cytK-1 positive presumptive B. cereus (4.4%). Generally, cytK-1 positive isolates belong to B. cytotoxicus species, which is not highly prevalent. In France, 5% of isolates among presumptive B. cereus were identified as the carriers of the cytK-1 gene, which is in line with our results [43]. Initially, cytK-1 was detected in the strain that induced several death cases [12] and for a long time it was considered that all carriers of this gene are highly cytotoxic. However, discoveries of the novel strains indicate that cytotoxicity among B. cytotoxicity is very diverse, and therefore should be further examined [44,45].
In the category of cereal products, including rice and seeds/pulses (n = 162), nheA carriers were found in 73.4%, hblA in 68.5%, while cytK-1 was found in 0.6% of examined products (129, 108, and 1/162, respectively). Potential emetic producers were detected in 20.37% (33/162) of tested products in this group. Specifically, in the subcategory of cereal grains, nheA was detected in almost 80% of presumptive B. cereus isolated from food (39/49). Buckwheat, millet, bulgur, quinoa, and rice were evidenced as a source of potentially toxinproducing B. cereus. Also, an experiment conducted on the flax grain produced in Canada showed that a large proportion of the B. cereus isolates carried the nheA (96.33%), hbID (94.5%), cytK-1 (12.8%), while only 2.8% strains carried ces gene [46], indicating that cereal grains are an important source of potentially toxin-producing B. cereus. Further, raw rice is usually highly contaminated with B. cereus. Samapundo et al. [47] reported that 100% of tested rice marketed in Belgium was contaminated by presumptive B. cereus. We noted that the majority of tested raw rice samples harbored at least one toxigenic B. cereus (27/29). nheA carriers were the most prevalent (26/29), followed by hblA (21/29) and emetic B. cereus (5/29). Berthod-Pluta et al. found that 81.4% of isolates obtained from rice were able to produce Nhe, 57.6% Hbl, and 6.8% were ces-positive [47]. In rice obtained from the U.S. market, 89.1% of B. cereus isolates possessed the nheAB genes [48], and almost 53.0% of tested isolates were able to produce both Nhe and Hbl enterotoxins. Overall, 93.9% of those isolates were determined as pathogenic [48], but emetic strains were not found.
Subcategory of cereal-based products and derivatives (n = 94) harbored nheA positive B. cereus in 70% (66/94) and hblA in 67% (63/94), respectively, while ces carriers were detected in 21.3% (20/94) of tested products. One sample in this group, namely couscous (n = 5), was contaminated with the all tested toxigenic B. cereus. Kone et al. [49] reported that only one cereal-based product (improved millet flour) out of 210 was contaminated with B. cytotoxicus, indicating that cereals do not represent the main source of B. cytotoxicus. The high prevalence of toxigenic B. cereus among cereals is not surprising. Cereals and cereal-based products (including seeds and rice) were determined as common sources of B. cereus food poisonings [1]. Nevertheless, cereals and cereal-based products are usually used as ingredients in mixed food or ready-to-eat products [24][25][26]31], indicating that they might be a significant contributory source of B. cereus. Several death cases were linked with cereulide production in ready-to-eat pasta and rice-based products [16,18,20,21,27]. Therefore, in this study, we also examined ready-to-eat products that contain pasta and rice. Based on the results obtained in our study, neither the ready-to-eat pasta nor ricebased products (n = 12) were contaminated with emetic strains. hblA and nheA positive B. cereus were found in two and 4/12 tested products, respectively, from which 2/12 we concomitantly contaminated with both toxin gene carriers. Yu et al. [50] reported that 35% of ready-to-eat products were positive for B. cereus, from which 39 and 83% of the isolated strains contained enterotoxin-encoding hblACD and nheABC genes, respectively, while 7% harbored the cesB gene. Similarly, in India, ready-to-eat products were mainly contaminated with nheABC genes (57.6%), followed by 36.8% of hblC positive B. cereus [51]. Ready-to-eat products are usually mild heat-treated, which enables bacterial spores to survive. These mild treatments inactivate the vegetative flora; however, they may trigger the germination of present spores. Depending on temperature growth affinities, intrinsic and extrinsic environmental factors, a growing population can produce cereulide or enterotoxins during refrigeration [15,52]. Furthermore, we tested instant soups (n = 13) constituted from starch and spices. Results showed that these products are largely contaminated with presumptive B. cereus (11/13). nheA, hblA, were identified in samples obtained from 11 soups, and ces was also found in six products. Similar observations were reported by Messelhäusseret et al. [53]; however, instant soups were not extensively examined in the past.
Milk powder has been a known source of B. cereus for many years [54,55], as the processing of milk into a powder does not eliminate B. cereus spores [56]. In our study, the toxin gene harboring presumptive B. cereus were highly prevalent in dairy-based powders. In total, 22/26 products were contaminated by the nheA, followed by hblA positive B. cereus (22/26). Although ces positive B. cereus s.l. are mainly related to starch-rich products, in 10 products belonging to dairy-based powders, potential cereulide producers were detected. Ten products from this group (10/26) were positive on nheA, hblA, and ces B. cereus carriers together. Liu et al. [57] reported that 75% of isolates were nha positive, 21% hbl positive, while ces encoding genes were not detected. Also, our study showed that other milk powder analogs, such as coffee creamers and chantilly cream represent the source of potentially toxin-producing B. cereus. One out of seven coffee creamers analyzed in this study were contaminated with nheA, hblA, and ces positive B. cereus together. In five products, nheA and hblA positive B. cereus were found combined, and one product was contaminated with the nheA gene carrier. Also, three products of chantilly cream obtained from the Belgian market were tested. Two products were contaminated in the combination of nheA, hblA, and ces positive B. cereus. Generally, the microbiological safety of dried milk powders depends on the initial level of contamination of raw milk, processing parameters, and hygiene in the production area. The main concern is related to reconstructed milk powder-based infant formulas, which in case of temperature abuse might cause infection in infants and babies due to the lack of competitive intestinal flora.
Dehydrated vegetable products such as mashed potato flakes are often contaminated with B. cereus s.l. In our study, nheA positive B. cereus were the most dominant contaminants of mashed potato flakes (n = 17). hblA positive B. cereus was found in nine products, ces carriers in seven products, and cytK-1 carriers were found in six products. Mashed potatoes flakes represent the most significant reported source of cytK-1 positive strains, considering that it has been rarely found in other products. Kone et al. [49] reported that 20/20 potato flakes were contaminated with B. cytotoxicus. Heini et al. [58] also found that mashed potato powder is the most significant source of the cytK-1 positive isolates. More considerably, our study showed that this product could be concomitantly contaminated with nhe, hbl, ces, and cytK-1 positive B. cereus together (2/17).
Spices (n = 12) and herbal teas (n = 8) also harbored potentially toxin-producing B. cereus. Among spices, 6/12 were contaminated with hblA positive B. cereus, while nheA positive B. cereus was found in 2/12 products. Six, four, and one tea product were contaminated with nheA, hblA, and ces carriers, respectively. Dry herbs and spices are well known to be contaminated with B. cereus. Indirectly, as ingredients in mixed food products, they can contribute to B. cereus food poisoning outbreaks and cases.

Growth of Toxigenic Presumptive B. cereus at 4, 7, and 10 • C
Psychrotrophic capacity varies within and among B. cereus species, as numerous lineages have been shown to grow at low temperatures. The principal food safety issue related to spore formers, such as B. cereus is the ability to survive the processing and preservation treatments and multiply during refrigerator storage to a concentration that might be infectious. Therefore in this study, we exposed toxigenic presumptive B. cereus communities obtained from different food products and ingredients (n = 121) to refrigerated (4 and 7 • C) and mild abuse temperatures (10 • C) for one month. In total, 94, 104, and 121/121 of presumptive B. cereus communities isolated from food products were able to grow at 4, 7, and 10 • C, respectively ( Table 2). As expected, at 4 • C growth detection time (GDT) was the longest. The GDT at 4 • C ranged from 1-23 days, with an average of 11 days and a median of 15 days. In the first five days, 16/94 of presumptive B. cereus were detected to grow. The GDT for 7/94 and 11/94 ranged between 6-10 days and 11-15 days, respectively. The majority of tested B. cereus (32/94) grew in the range of 16-20 days, while for 18 tested B. cereus, the GDT was between 20-25 days. Day 23 was the last day when B. cereus was detected at 4 • C. It was not possible to determine a correlation between specific food groups and the presence of psychrotrophic presumptive B. cereus as psychrotrophs were highly prevalent in all food categories. In the category of cereal products, including rice and seeds/pulses, B. cereus was detected in a total of 48/70 of tested samples. Food products typically linked with B. cereus severe food poisonings, such as pasta and rice were also a source of psychrotrophic B. cereus (6/11 and 6/9, respectively). B. cereus obtained from powdered dairy-based products could also grow at 4 • C (7/7). Psychrotrophic presumptive B. cereus communities were found in coffee creamer (6/8) and chantilly cream (3/3), indicating that milk powder analogs are also a great source of psychrotrophic B. cereus able to grow at refrigerator temperature. Also, other products showed as a potential source of toxigenic presumptive B. cereus that can grow at refrigerator temperatures, such as mashed potato flakes (9/10), instant soups (12/12), and spices (6/7).
Although our study demonstrated that psychrotrophic B. cereus are highly prevalent among different food products, data from the literature is diverse. For example, Samapundo et al. [47] showed that among 380 isolates, none were able to grow at 5 • C in 45 days. Similar results were reported by Park et al. who isolated B. cereus from the green lettuce [59]. Isolates obtained from cooked chilled products did not grow at 4 • C; however, 10% of these strains were able to grow at 5 • C [60]. Carlin et al. reported that 9/83 of isolates obtained from food and environment were able to grow at 4 • C [61]. B. cereus obtained from condiments were all psychrotrophic [62]. Currently, there is no standardized assay for defining B. cereus temperature growth affinities. Factors such as selection of media, growth conditions (i.e., temperature, agar plate vs. broth, shaking vs. non-shaking) represent potential sources of variation among data. While some researchers have suggested usage of the "psychrotolerant signatures" obtained via cold shock protein (encoded by cspA) or panC, this approach is still questioned as some non-psychrotolerant strains also encode these "psychrotolerant signatures" or are able to grow at low temperatures but do not possess any of the genes that would indicate there temperature growth affinities [63,64]. Based on the differences in 16 s rRNA, Wijnands et al. [30] reported that 4.4% of food isolates were psychrotrophic. Furthermore, researchers estimated psychrotrophic profiles by exposure of isolates at 6 • C. Isolates obtained from food products in Morocco had ability to grow at 6 • C (37/52) [65]. Also, Stenfors and Granum found that 17/26 were able to grow at 6 • C among dairy, food, and clinical isolates [66]. Jan et al. [64] reported that all isolates from egg products multiplied at 6 • C. Similarly, Torkar and Seme [67] detected 56.7% of B. cereus food and clinical isolates at 6.5 • C.
At 7 • C, 104/121 of presumptive B. cereus were detected. The GDT range and average were similar to results obtained at 4 • C (1-22 and 10 days, respectively); however, the median value was lower (11 days). In the first five days, 28/104 of tested B. cereus were detected. After 6-10 and 11-15 days, 15 and 21/104 of B. cereus were observed. The highest number of B. cereus was able to grow in the range between 16-20 days (34/104), while 6/104 were detected after 20 days of exposure at 7 • C. In the category of cereal products, including rice and seeds/pulses, B. cereus was detected in 58/70. Specifically, colonies from rice and pasta were detected in 7/8 and 8/10 of tested products. Further, presumptive B. cereus obtained from milk powder and powders containing milk or whey protein had the same ability to grow at 7 • C as at 4 • C. For colonies obtained from mashed potato flakes, instant soups, seeds, and herbs, and spices growth potential at 7 • C was slightly higher (10/10, 12/12, and 7/7, respectively). Berthold-Pluta et al. [38] tested the psychrotrophic properties of 1022 isolates obtained from different food products at 7 • C for 10 days. Results showed that 25% (256) isolates were able to grow at 7 • C, which is in line with our study where 21/104 (20.2%) grew at 7 • C in 10 days. However, the majority of tested B. cereus communities started to grow after 10 days. Exposure time in our study was longer; therefore we could observe more psychrotrophs. Similar results were reported for isolates obtained from refrigerated dairy products. Four out of 15 isolates (26.7%) were detected at 7 • C after 10 days of exposure. Carlin et al. [61] reported that 41/83 (49%) diarrheal and food-environment strains grew at 7 • C after 12 days of exposure. Among the 101 isolated B. cereus from green lettuce, only 18 were capable of growing at 7 • C [59].
Results obtained at 10 • C indicate that all presumptive B. cereus exhibit mesophilic character considering that 121/121 were able to grow at this temperature. On the 16th day, presumptive B. cereus were detected in all tested samples, with an average of seven days and a median value of four days. For the near half of the inoculated agar plates (45%), the GDT was in the first five days. After, 21 and 19/121 were detected to grow between 6-10 and 11-15 days. Other studies also reported that many B. cereus can grow at 10 • C. Samapundo et al. [47] detected the majority of the isolates (87.9%) during 45 days of exposure. Isolates obtained from dairy products also displayed psychrotrophic character (12/15) [68].
Temperature profiling of food isolates represents an important factor for food safety and quality. Determination of potential growth or toxin production at a given temperature may provide information about behavior in a specific food product that is directly related to food storage recommended temperature. Especially, spore formers are a significant risk in the case of temperature/time storage abuse. Although they grow very slowly at 4 and 7 • C; at 10 • C, psychrotrophs can grow fast and may produce toxins.

Toxigenic Profiles of Psychrotrophic Presumptive B. cereus Grown at 4 • C
Several studies have shown that psychrotrophic B. cereus carry toxin encoding genes associated with both diarrheal and emetic disease. To assess the potential hazard of psychrotrophic presumptive B. cereus that are able to grow at refrigerated temperatures (n = 94), their toxigenic profiles were determined. Enterotoxin and cereulide encoding genes were detected in 81% (76/94) of psychrotrophic presumptive B. cereus biomasses isolated from food. The nheA gene was the most prevalent at 70.2% (66/94), followed the by hblA gene at 55% (52/94), and the ces encoding gene at 11.7% (11/94). None of the psychrotrophic presumptive B. cereus were cytotoxin K-1 encoding gene (cytK-1) positive. The main carrier of this gene is B. cytotoxicus species, which has been characterized by thermotolerant growth affinities. Presumably, ribosomes in thermophilic bacteria are nonfunctional at lower temperatures [69]; therefore, the absence of this microorganism among psychrotrophs was expected. Park et al. [59] reported that 94% of the psychrotolerant B. cereus isolates obtained from green lettuce harbored the nheABC genes, 44% hbl, while ces was not detected. Similarly, Bartoszewicz et al. [70] reported that 85 and 30% of psychrotrophic isolates were nheA, hblA positive, respectively. Together, hblA and nheA were present in 40% (38/94) of B. cereus communities that were able to grow at 4 • C, while the combination of hblA and ces was not noted. hblA, nheA, and ces psychrotrophic presumptive B. cereus were found together in 5.3% (5/94) of tested food products. Our data are consistent with previous reports demonstrating that enterotoxin encoding genes are prevalent among psychrotrophic B. cereus. Simultaneously, ces and nheA were detected in 7.44% (7/94), while ces was found alone only in one sample. Frequency distributions of the growth detection time (GDT, day) of enterotoxigenic and emetic psychrotrophic presumptive B. cereus are shown in Figure 1.
Toxins 2022, 14, x FOR PEER REVIEW 9 of 16 for many years due to discrepancies among data. Investigations related to foodborne outbreaks did not report properly chilled food as the main cause of B. cereus poisoning [29]. At low temperatures, B. cereus proliferates slowly; however, the effects of the food matrix seem to play an important role in toxin production.

Cereulide Production by Psychrotropic Presumptive B. cereus at 37 °C and 4 °C
Cereulide producers are characterized by mesophilic and psychrotrophic growth capacities [68]. Psychrotrophs mainly belong to the B. weihenstephanensis species. However, the potential for psychrotrophic B. cereus group strains to acquire the ces genes by horizontal gene transfer exists. Therefore it has been assumed that potential cereulide psychrotrophic might not all belong strictly to this species [29]. In our study, we identified According to results obtained in this study, mashed potato flakes seem like the significant carriers of psychrotrophic enterotoxigenic B. cereus. Five out of nine psychrotrophic presumptive B. cereus were both hblA and nheA positive, while one out of nine was only nheA positive. Similarly, instant soups showed as a great source of toxigenic psychrotrophic B. cereus considering that 10/12 psychrotrophic communities were positive on some of the tested genes. Eight out of twelve were hblA and nheA positive, one out of twelve was ces and nheA positive. In one sample nheA positive psychrotrophic B. cereus were detected. Further, herbs and spices were contaminated with enterotoxigenic B. cereus (five out of six). While not many studies were conducted to examine toxigenic properties of psychrotrophic B. cereus isolated from specific food groups, concerns have been raised for many years due to discrepancies among data. Investigations related to foodborne outbreaks did not report properly chilled food as the main cause of B. cereus poisoning [29]. At low temperatures, B. cereus proliferates slowly; however, the effects of the food matrix seem to play an important role in toxin production.

Cereulide Production by Psychrotropic Presumptive B. cereus at 37 • C and 4 • C
Cereulide producers are characterized by mesophilic and psychrotrophic growth capacities [68]. Psychrotrophs mainly belong to the B. weihenstephanensis species. However, the potential for psychrotrophic B. cereus group strains to acquire the ces genes by horizontal gene transfer exists. Therefore it has been assumed that potential cereulide psychrotrophic might not all belong strictly to this species [29]. In our study, we identified potential cereulide producers that can grow at refrigerator temperatures with the aim to examine the ability to produce cereulide. Presumptive psychrotrophic B. cereus identified as ces gene positive were tested by the computer-aided semen analysis study of the boar semen motility described by Rajkovic et al. [69]. After exposure to 4 • C, 11 out of 38 examined ces positive B. cereus were detected to grow. These B. cereus were isolated from the following food products: buckwheat, chantilly cream, coffee creamer, couscous, milk powder, muesli, polenta, instant vermicelli soup, and instant meat soup (Table 3).
Based on the motility assay, it was seen that 5/11 of ces-positive presumptive B. cereus were able to produce cereulide at 37 • C. Our results are consistent with the study of Biest-Pieters et al. [39], who reported that emetic strains do not always display their pathogenic potential. Food products from which psychrotophic cereulide producers were obtained are buckwheat, chantilly cream, coffee creamer, muesli, and instant vermicelli soup. Further, to estimate the ability to produce cereulide in small quantities at 4 • C, we used LC-MS 2 . None of the ces-positive samples were able to produce any significant amounts of cereulide at 4 • C after one month of exposure. Table 3. GDT and cereulide production at 37 and 4 • C of psychrotrophic ces-positive B. cereus.  Only in one sample 0.79 ng/mL of cereulide was quantified. Limited data are available about cereulide production at refrigerator temperatures. It has been noted that at 8 • C emetic strains produce minimum amounts of toxin [29,71]. This can explain why correctly stored products were never reported as a cause of intoxication with cereulide. However, it should not be ignored that, in many cases, food products are subjected to a temperature that often surpasses recommended refrigerator temperatures and, in numerous cases, can be higher than 10 • C. Cereulide synthesis is a multifactorial process that is not strictly dependent on the cell number. Present nutrients can trigger production even before the exponential growth phase [72]. Moreover, cereulide at low temperatures can be produced in high amounts [73] or in more potent forms (isocereulide A) [74,75].

Conclusions
This study demonstrated that selected food ingredients and products were significantly contaminated with toxigenic B. cereus. Many of them were concomitantly contaminated with different potentially pathogenic B. cereus gene carriers, indicating that one product may pose a risk for both diarrheal and emetic disease. The relatively high number of isolated psychrotrophic presumptive B. cereus were enterotoxin and cereulide gene carriers, indicating that refrigeration cannot ensure complete growth prevention. Due to the increasing number of foodborne outbreaks caused by B. cereus toxins, it is necessary to monitor the presence of this pathogen among food products more regularly. Moreover, in order to comply with an appropriate level of consumers protection, relevant parties such as food business operators and health authorities must take into account risks present during food handling. Since toxigenic psychrotrophic B. cereus is abundant in food products and ingredients, it is important to estimate possible risks related to the refrigerator and temperature abuse conditions in regards to both enterotoxin and cereulide producing B. cereus. Therefore, additional studies on factors that affect B. cereus growth and toxin production are needed.

Collection of Food Products
A total of 250 food products were collected from Belgian, Dutch, and Serbian retail markets. Selected products are mainly used as ingredients in mixed food products (n = 250). Products were classified into five categories: 'cereal products, including rice and seeds/pulses', 'dairy-based products', 'dehydrated vegetables, 'mixed food', 'herbs and spices' according to the Manual for reporting on food-borne outbreaks in accordance with Directive 2003/99/EC published by EFSA (Table 4) [76]. Individual products within group were selected from different brands available in retail shops during 2019 and stored at recommended conditions prior to analysis. Analysis was performed a maximum of one day after purchase of ready-to-eat products and sprouted seeds or five days upon arrival of dry food products in the Laboratory of Food Microbiology and Food Preservation (Ghent University). Oat flakes 5 Flour 13 Pasta and pasta-like products 50 Seeds 7 Sprouted seeds 12

Detection of Presumptive B. cereus
Detection of presumptive B. cereus was performed as previously described [47]. In brief, 25 g of selected food product was placed in a sterile filter stomacher and enrichment broth composed of Tryptone Soy Broth (TSB) (Oxoid, UK) and 50,000 IU of polymyxin B supplement (Oxoid, Hampshire, UK) was added to the total of 250 g. Enrichment of homogenized samples was performed at 30 • C for 24 h, after which 0.1 mL was inoculated onto a mannitol-egg yolk-phenol-red polymyxin-agar medium (Oxoid, Hampshire, UK), supplemented with polymyxin B according to manufacturer instructions and incubated at 30 • C for 48 h.

Toxin Gene Profiling
DNA isolation was performed using Prepman lysis solution (Applied Biosystems, Waltham, MA, USA). Fresh culture of presumptive B. cereus was collected and placed in a lysis buffer in 2 mL tube, mixed thoroughly, and incubated at 95 • C for 15 min. Afterwards, supernatants with isolated DNA were stored at −20 • C and screened for the presence of enterotoxin and cereulide encoding genes using primers as previously described (Table 5), namely the nheA, hblA, cytK-1, and cereulide toxin-related genes ces. The specificity of used primers was primarily tested in Basic Local Alignment Search Tool Genomic (https://blast.ncbi.nlm.nih.gov/Blast.cgi (5 April 2019). Further, they were checked by testing 30 reference strains with known toxigenic properties obtained from the culture collection of Laboratory of Food Microbiology and Food Preservation (Ghent University). Genomic DNA extracted from strains ATCC 14579, DSM 4384, NVH 1230-88, NCTC 11145, NS115, NS117, LMG 12334, F4346/75, NVH0075/95, NVH0500/00, F0285/78 and NVH0391-98 were used as positive or negative controls in this study (also for cereulide production in Section 4.5), depending on their toxigenic properties, while the blank control was done using nuclease-free water.
A typical 50 µL PCR mixture for the SYBR green I real-time PCR assay consisted of 2 µL of template DNA, 25 µL of 2x concentrated PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania), 500 nM of reverse and forward primers (IDT Technologies, Leuven, Belgium) and 18 µL of nuclease-free water (Ambion, Austin, TX, USA). Primarily, isolated DNA was exposed to 95 • C for 10 min, followed by 40 cycles of 95 • C/15 s for denaturation, and annealing and extension at temperatures shown in Table 5. In the subsequent dissociation stage for the analysis of melting curve, the temperature was raised from 60 • C to 95 • C. Further, 20 µL PCR reaction mixture for the probe-based assay contained 10 µL of GoTaq Probe qPCR (Promega, Madison, WI, USA), 500 nM of each primer, 200 nM probe, and 2 µL of the template DNA. All PCR reactions were performed and analyzed in ABI 7300 Real Time PCR system (Applied Biosystems, Waltham, MA, USA) using MicroAmp 96-well plates (Applied Biosystems, Waltham, MA, USA). For the ces gene detection, two pair of primers were used (Table 5).

Refrigeration and Mild Abuse Temperature Exposure
Collected presumptive B. cereus communities, which possessed at least one virulence gene, were tested for the capability to grow at psychotropic conditions at 4, 7, and mild abuse temperature (10 • C). Prior to exposure to respective temperatures, cultures were reactivated in TSB. After, culture was transferred onto double Tryptone Soya Agar (TSA) and incubated for 30 days at 4, 7, and 10 • C.

Emetic Toxin Detection via Boar Semen Motility Assay
The procedure was performed according to Rajkovic, Uyttendaele, and Debevere [80]. Primarily, ces-positive B. cereus samples were incubated for 24 h at 37 • C, in TSB after which 1 mL of each sample was inoculated onto TSA (in triplicates), air-dried and incubated again at 30 • C for 24 h. Grown colonies were flooded with 3 mL of methanol (≥99.9%, Sigma-Aldrich, St. Louis, MI, USA) and transferred to the 500 mL Erlenmeyer flask. Methanol was added to the final dilution of 1:3 of biomass and placed in a water bath at 100 • C for 1 h. Extracted cereulide was stored in glass vials at −20 • C until analysis. Further, five microliters of cereulide extract were mixed with 195 µL of the fresh boar semen (ca. 30 million cells per mL) in a microtiter well, and then 5 µL of suspension was immediately transferred into Leja Slides (2-chambered 20 mm slide, Leja, Nieuw-Vennep, The Netherlands). The sample was considered cereulide-positive when interruption of semen motility occurred within less than 10 min. Motility was microscopically analyzed (Zeiss, AxioCam Mrm Imager A.1, Jena, Germany) with a MiniTherm stage warmer providing an optimal temperature of 37 • C. The images were examined via the AxioVision program. Experiments were performed in three repetitions. 4.6. Quantification of Cereulide Production at 4 • C by LC-MS 2 Analysis ces positive samples that were able to grow at 4 • C were tested for the cereulide production at 4 • C by LC-MS 2 . After one month of exposure, we extracted cereulide as previously described (Section 4.5.) and subjected to further analysis according to Delbrassinne et al. [81]. Briefly, the LC-MS LCQ Deca-XP Plus ion trap mass analyzer (ThermoFinnigan, San Jose, CA, USA) was used for analysis. A full mass spectrum from 500 to 1300 m/z values and an MS 2 fragmentation mass spectrum were recorded in positive electrospray mode (ESI+). The peaks corresponding to the loss of a CO (m/z values 1125.3 and 1083.3, respectively) were always the highest peaks generated through the fragmentation. The chromatograms were smoothed thanks to a Gaussian function.